Dit protocol maakt de bereiding en karakterisering mogelijk van CD47- en integrine α4/β1-co-gemodificeerde macrofagen, membraan-gecoate poly(melkzuur-co-glycolzuur) nanodeeltjes voor gerichte toepassingen van geneesmiddelafgifte.
Method Article
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 7-Aminoactinomycin D (7-AAD) | Thermo Fisher Scientific (USA) | A1310 | Viability dye for flow cytometry live/dead discrimination |
| Alexa Fluor 488-conjugated goat anti-rabbit IgG | Thermo Fisher Scientific (USA) | A-11008 | Secondary antibody for CD68 immunofluorescence staining |
| Anti-mouse CD47 antibody | Abcam (USA) | ab214453 | Used for Western blot analysis to verify CD47 expression |
| Anti-mouse integrin α4 antibody (B-2) | Santa Cruz Biotechnology (USA) | sc-376334 | Used for Western blot analysis to verify integrin α4 expression |
| Anti-mouse integrin β1 antibody (A-4) | Santa Cruz Biotechnology (USA) | sc-374429 | Used for Western blot analysis to verify integrin β1 expression |
| ApoE-/- mice | The Jackson Laboratory (USA) | N/A | Used for in vivo atherosclerosis model |
| BCA assay kit | Thermo Fisher Scientific (USA) | 23225 | Used to determine protein concentration |
| Black silicone plate | N/A | N/A | Used to mount aorta for ex vivo imaging |
| CD47 plasmid (pcDNA3.1(+)) | Generay Biotech (China) | G0177361-1 | Contains mouse Cd47 gene for overexpression |
| Colchicine | Sigma-Aldrich (USA) | C9754 | Therapeutic payload in nanoparticles |
| Confocal laser scanning microscope | Leica Microsystems | TCS SP8 | Used for fluorescence imaging and co-localization analysis |
| Citrate buffer (pH 6.0) | Thermo Fisher Scientific (USA) | N/A | Used for antigen retrieval in immunofluorescence staining |
| Dialysis tubing (MWCO 3,500 Da) | Sigma-Aldrich (USA) | PURD35030 | Used for nanoparticle purification |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich (USA) | D2447 | Solvent for nanoparticle preparation |
| DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) | Beyotime (China) | C1036 | Fluorescent labeling of nanoparticle core |
| DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) | Beyotime (China) | C1038 | Fluorescent labeling of macrophage membrane |
| DiR | Beyotime (China) | Y239280 | Used for in vivo fluorescence imaging |
| DAPI | Thermo Fisher Scientific (USA) | D3571 | Nuclear counterstain for fluorescence imaging |
| Dulbecco’s Modified Eagle Medium (DMEM) | Thermo Fisher Scientific (USA) | 11965092 | Cell culture medium |
| Dynamic light scattering analyzer (Zetasizer Nano ZS) | Malvern Panalytical (UK) | N/A | Used for dynamic light scattering (DLS) measurements of nanoparticle size, PDI, and zeta potential |
| ELISA kit | R&D Systems (USA) | Depends on target cytokine | Used to quantify inflammatory cytokines |
| Endothelin-1 (ET-1) | Sigma-Aldrich (USA) | E7764 | Used to stimulate integrin α4/β1 expression |
| Fetal bovine serum (FBS) | Gibco (Thermo Fisher Scientific, USA) | 10091148 | Supplement for cell culture medium |
| Flow cytometer | BD Biosciences (USA) | FACSCalibur | Used for fluorescence signal acquisition |
| Flow cytometry analysis software (FlowJo v10.8.1) | BD Biosciences (USA) | N/A | Used for flow cytometry data analysis |
| Human umbilical vein endothelial cells (HUVECs) | FuHeng Biotechnologies (China) | FH1122 | Used for in vitro targeting assays |
| Image analysis software (ImageJ) | National Institutes of Health (USA) | N/A | Used for quantitative image analysis |
| In vivo imaging system | PerkinElmer (USA) | IVIS Lumina III | Used for whole-body fluorescence imaging |
| Isopropanol (60%) | Sigma-Aldrich (USA) | 34863 | Used for differentiation during Oil Red O staining |
| Lipofectamine 3000 | Thermo Fisher Scientific (USA) | L3000001 | Transfection reagent |
| Magnetic stirrer and stir bar | N/A | N/A | Used for nanoparticle preparation |
| Masson’s Trichrome stain kit | Abcam (USA) | ab150686 | Used for collagen staining |
| Mini extruder | Avanti Polar Lipids (USA) | 610000 | Used for membrane extrusion |
| Murine RAW 264.7 cell line | FuHeng Biotechnologies (China) | FH0328 | Source of macrophages |
| Murine smooth muscle cell line | FuHeng Biotechnologies (China) | FH-Y063 | Used for safety evaluation |
| Oil Red O | Sigma-Aldrich (USA) | O0625 | Used for lipid staining |
| Opti-MEM | Thermo Fisher Scientific (USA) | 31985062 | Reduced-serum medium for transfection |
| Oxidized low-density lipoprotein (Ox-LDL) | Yiyuan Biotechnologies (China) | YB-002 | Used to induce foam cell formation |
| Penicillin–streptomycin | Thermo Fisher Scientific (USA) | 15140122 | Antibiotic supplement for cell culture |
| Phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich (USA) | 78830 | Protease inhibitor |
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific (USA) | 10010023 | Washing and dilution buffer |
| Poly(lactic-co-glycolic acid) (PLGA, 50:50; MW 90,000) | Dalian Meilun Biotechnology (China) | MB5649-1 | Polymer for nanoparticle fabrication |
| Polycarbonate membrane filters (200 nm, 400 nm) | Whatman (UK) | 800282 | Used for extrusion |
| Potassium chloride (KCl) | Sigma-Aldrich (USA) | P9333 | Component of lysis buffer |
| P3000 reagent | Thermo Fisher Scientific (USA) | L3000001 | Transfection enhancer |
| Rotary microtome | Leica Biosystems (Germany) | RM2235 | Used for paraffin sectioning |
| Syringe pump | Harvard Apparatus (USA) | 70-3007 | Used for controlled nanoparticle formation |
| Transmission electron microscope (TEM) | Thermo Fisher Scientific (USA) | Talos F200S | Used for nanoparticle imaging |
| Tris-HCl | Sigma-Aldrich (USA) | T2663 | Buffer component |
| Tumor necrosis factor alpha (TNF-α) | R&D Systems (USA) | 410-MT | Used to activate endothelial cells |
| Ultrapure water | Milli-Q (Merck, USA) | ZMQSV0T01 | Used for nanoparticle preparation and dialysis |
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